Methods and compositions for nuclease-mediated targeted integration

ABSTRACT

Disclosed herein are methods and compositions for targeted, nuclease-mediated insertion of transgene sequences into the genome of a cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/630,128, filed Feb. 24, 2015, which claims the benefit ofU.S. Provisional Application No. 61/943,865, filed Feb. 24, 2014, thedisclosure of which are hereby incorporated by reference in theirentireties.

TECHNICAL FIELD

The present disclosure is in the fields of gene modification andincreasing targeted integration of exogenous sequences into the genomeof a cell.

BACKGROUND

Engineered nucleases, including zinc finger nucleases, TALENs,CRISPR/Cas nuclease systems, Ttago nucleases and homing endonucleasesdesigned to specifically bind to target DNA sites are useful in genomeengineering. For example, zinc finger nucleases (ZFNs) and TALENs(including TALENs comprising FokI-TALE DNA binding domain fusions, MegaTALs and cTALENs) are proteins comprising engineered site-specific zincfingers or TAL-effector domains fused to a nuclease domain. Suchnucleases have been successfully used for genome modification in avariety of different species at a variety of genomic locations. See, forexample, See, e.g., U.S. Pat. Nos. 8,623,618; 8,034,598; 8,586,526;6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,067,317; 7,262,054;7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; U.S.Patent Publications 20030232410; 20050208489; 20050026157; 20060063231;20080159996; 201000218264; 20120017290; 20110265198; 20130137104;20130122591; 20130177983, 20130177960 and U.S. Pat. No. 9,873,894, thedisclosures of which are incorporated by reference in their entiretiesfor all purposes.

Cleavage of a target nucleotide sequence by these nucleases increasesthe frequency of homologous recombination (HR) with a donor at thetargeted locus by more than 1000-fold. Homology-directed repair (HDR) ofa nuclease-mediated cleavage event can be used to facilitate targetedinsertion of a gene (transgene) by co-delivering a donor moleculeencoding a gene flanked by sequence homologous to region surrounding thebreak site. In addition, the repair of a site-specific DSB bynon-homologous end joining (NHEJ) can also result in gene modification,including gene (transgene) insertion by NHEJ-dependent end capture. See,e.g., U.S. Patent Publication No. 20110207221. In addition to targetedintegration of a transgene, nuclease-mediated cleavage and repair byNHEJ can result in non-specific insertions and/or deletions (“indels”)at the site of the break. Thus, nucleases specific for the targetedregion can be utilized such that the transgene construct is inserted byeither HDR- or NHEJ-driven processes, or for knockout of a gene througherror-prone NHEJ repair of the nuclease-mediated DSB. Gene correctionmay also be accomplished using targeted nucleases and donor moleculesdesigned to replace a specified region in an endogenous gene withsequences supplied in the donor. A specific double strand break (DSB) isintroduced in the gene and in the presence of the gene correcting donorDNA, the sequences of interest are replaced using those of the donor viahomology dependent recombination.

This nuclease-mediated targeted transgene insertion approach offers theprospect of improved transgene expression, increased safety andexpressional durability, as compared to classic integration approaches,since it allows exact transgene positioning to minimize the risk of genesilencing or activation of nearby oncogenes. However, efficiency ofnuclease activity can be influenced by a variety of factors such asaccessibility of the chromosomal DNA target and the quality of thebinding interaction between the nuclease and its target nucleic acid.Efficiency of these approaches in vivo is further complicated by factorssuch as target tissue accessibility and tissue uptake of vectors thatdeliver the nucleases and transgene donors, and nuclease expressionlevels that can be achieved in vivo. To increase the success rate ofnuclease driven genomic modifications, researchers often have to resortto introducing selectable markers during donor integration in order tobe able to select variants that have had modifications from those thathave not been modified (see, for example, U.S. Pat. No. 6,528,313). Fora number of applications, use of selectable markers is not desirable asthis technique leaves an additional gene or nucleic acid sequenceinserted into the genome.

Thus, there remains a need for compositions and methods for increasingnuclease-mediated targeted integration of transgenes to allow for evenmore efficient use of these powerful tools.

SUMMARY

Disclosed herein are methods and compositions for nuclease-mediatedintegration of one or more exogenous sequences into a target sequencevia sequential administration of the nuclease(s) and the exogenoussequences(s). The methods and compositions described herein increase theefficiency of nuclease-mediated targeted integration of exogenoussequences (transgenes). In particular, the methods and compositionsinvolve sequential separate administration of nucleases and transgenes,for example, administration of separate solutions of nuclease(s) andtransgene(s) with a delay between the separate administrations. Thedelay between administrations may be minutes, hours or days or evenlonger, for example, 10 minutes or more, 30 minutes or more, 1 hour ormore, 2 hours or more, 3 hours or more, 24 hours or more, 36 hours ormore, 48 hours or more, 72 hours or more, or 4 days or more, 5 days ormore, 6 days or more, a week or more, or even longer betweenadministrations. The methods and compositions described herein result inan enhanced efficiency of transgene integration (e.g., an increase of10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10 to 100 fold(or any value therebetween) or even more in transgene integration) ascompared to transgenes integrated using alternate methods (e.g.,concurrent administration or serial administrations less than at least 4hours apart).

Thus, in one aspect, described herein is a method of integrating anexogenous sequence into a target sequence of an isolated cell, themethod comprising sequentially administering (i) one or more nucleasesthat cleave the target sequence and (ii) one or more donor sequencesthat are integrated into the target sequence following cleavage of thetarget sequence by the one or more nucleases, wherein there is a delayof at least 24 hours between the sequential administration. In certainembodiments, the delay between the sequential administrations is between24 and 72 hours. In one embodiment, the one or more nucleases areadministered prior to the one or more donors and the one or morenucleases and/or one or more donors are administered using a plasmid, aviral vector (e.g., AAV vector) or in RNA, mini-circle or linear DNAform. In another embodiments, the one or more donors are administeredprior to the one or more nucleases and the one or more nucleases areadministered in RNA form (e.g., in mRNA form).

Nucleases, for example engineered meganucleases, zinc finger nucleases(ZFNs), TALE-nucleases (TALENs including fusions of TALE effectorsdomains with nuclease domains from restriction endonucleases and/or frommeganucleases (such as mega TALEs and compact TALENs)) Ttago nucleasesand/or CRISPR/Cas nuclease systems are used to cleave DNA at anendogenous locus (e.g. safe harbor gene or locus of interest) in thecell into which any exogenous donor sequence (transgene) is inserted.Targeted insertion of a donor transgene may be via homology directedrepair (HDR) or non-homology repair mechanisms (e.g., NHEJ-mediated endcapture). Insertions and/or deletions (“indels”) of nucleotides (e.g.,endogenous sequences) may also occur at the site of integration. Thenuclease can induce a double-stranded (DSB) or single-stranded break(nick) in the target DNA. In some embodiments, two nickases are used tocreate a DSB by introducing two nicks. In some cases, the nickase is aZFN, while in others, the nickase is a TALEN or a CRISPR/Cas system.

In one aspect, the methods comprise administering one or more nucleasesto a cell (e.g., one or more vectors encoding the nucleases) such thatthe vectors comprising the encoded nucleases are taken up by the cell,then the nucleases cleave a specified endogenous locus of the cell'sgenome and finally, after a period of time, administering one or moreexogenous (donor) sequences to the cells (e.g. one or more vectorscomprising these exogenous sequences) such that the exogenous sequencesare integrated (in a targeted manner) at or near the cleaved genome(e.g., the nuclease(s) binding and/or cleavage site(s)), for example,within 1-300 (or any value therebetween) base pairs upstream ordownstream of the site(s) of cleavage, more preferably within 1-100 basepairs (or any value therebetween) of either side of the binding and/orcleavage site(s), even more preferably within 1 to 50 base pairs (or anyvalue therebetween) on either side of the binding and/or cleavagesite(s). The exogenous sequence may be administered any time afteradministration of the nucleases, for example, anywhere from 10 minutesor more, 30 minutes or more, 1 to 72 hours or more (4 days, 5 days, 6days, 7 days or more). In certain embodiments, the period of timebetween administration of the nuclease(s) and donor is between 24 hoursand 4 days, preferably 48-72 hours. In certain embodiments, the cell isan isolated cell and is cultured between administration of thenuclease(s) and administration of the donor transgene.

In another aspect, the methods comprise administration of an exogenoussequence (e.g. a vector, plasmid, mini-circle or linear DNA comprisingthe exogenous sequences) to a cell followed by administration of thenuclease (e.g. administration of an mRNA encoding the nuclease, vectoror plasmid encoding the nuclease, or administration of the nuclease asits protein form). For example, the mRNA encoding the nuclease, or theprotein nuclease may be administered any time after the exogenoussequence, for example, anywhere from 10 minutes or more, 30 minutes ormore, 1 to 72 hours or more (4 days, 5 days, 6 days, 7 days or more). Incertain embodiments, the period of time between administration of thedonor and the nuclease(s) is between 24 hours and 4 days, preferably48-72 hours.

In some embodiments the nuclease(s) is/are administered as an RNA (e.g.,as their encoding mRNAs). In some embodiments, the mRNA comprises thetwo nucleases of a nuclease pair, separated by a ribosomal stutteringsite, and internal ribosome entry site or the like (e.g. a 2A sequenceor IRES). In other embodiments, two mRNAs encoding the two nucleases ina nuclease pair are separate mRNAs which can be combined before orduring administration to the cell. In some embodiments, the mRNAs aremodified or capped.

Cleavage can occur through the use of specific nucleases such asengineered zinc finger nucleases (ZFNs), transcription-activator likeeffector nucleases (TALENs), using Ttago nucleases or using theCRISPR/Cas system with an engineered crRNA/tracr RNA (‘single guideRNA’) to guide specific cleavage. In some embodiments, two nickases areused to create a DSB by introducing two nicks. In some cases, thenickase is a ZFN, while in others, the nickase is a TALEN or aCRISPR/Cas system. Targeted integration of exogenous donor sequences mayoccur via homology directed repair mechanisms (HDR) and/or vianon-homology repair mechanisms (e.g., NHEJ-mediated end capture). Thenucleases as described herein may bind to and/or cleave the region ofinterest in a coding or non-coding region within or adjacent to thegene, such as, for example, a leader sequence, a regulatory sequence,trailer sequence or intron, or within a non-transcribed region, eitherupstream or downstream of the coding region. In certain embodiments, thenuclease cleaves the target sequence at or near the binding site.

In any of the methods described herein, the donor sequence may compriseone or more transgenes that express protein products. In certainembodiments, the protein products are therapeutic in that they arefunctional versions of proteins aberrantly expressed in a disorder(e.g., a genetic disorder such as a hemophilia, lysosomal storagediseases, metabolic diseases, hemoglobinopathies and the like). Incertain embodiments, the transgene encodes one or more functionalclotting factor proteins (e.g., Factor VII, Factor VIII Factor IX and/orFactor X). In some embodiments, the donor sequence is designed tocorrect a mutation in an endogenous gene via nuclease-dependent HDR.

The nuclease may target any endogenous locus. In certain embodiments,the transgene is integrated in a site-specific (targeted) manner usingat least one nuclease (e.g., ZFNs, TALENs and/or CRISPR/Cas systems)specific for a safe harbor locus (e.g. CCR5, HPRT, AAVS1, Rosa oralbumin. See, e.g., U.S. Pat. Nos. 7,888,121; 7,972,854; 7,914,796;7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S. Patent Publications20030232410; 20050208489; 20050026157; 20060063231; 20080159996;201000218264; 20120017290; 20110265198; 20130137104; 20130122591;20130177983, 20130122591, 20130177960 and U.S. Pat. No. 9,873,894).

In another aspect, described herein is a method of genetically modifyinga cell to comprise an exogenous sequence, the method comprising cleavingan endogenous gene in the cell using one or more nucleases (e.g., ZFNs,TALENs, CRISPR/Cas) and, after a period of time, administering thetransgene to the cell such that it is integrated into the endogenouslocus and expressed in the cell. In certain embodiments, the endogenousgene is a safe harbor gene such as CCR5, HPRT, AAVS1, Rosa or albuminlocus.

In another aspect, described herein is a method of genetically modifyinga cell to comprise one or more exogenous sequences, the methodcomprising administering a vector comprising the exogenous sequence tothe cell, allowing sufficient time for uptake of the vector comprisingthe exogenous sequence by the cell, and then administration of mRNAsencoding one or more nucleases (e.g. ZFNs, TALENs (TAL-effector domainsand nuclease domains (restriction endonucleases and/or meganuclease))and/or a CRISPR/Cas system) or administering nucleases as proteins, suchthat the nucleases cleave the endogenous gene and the exogenoussequences are integrated and expressed. In certain embodiments, theendogenous locus is a safe harbor locus such as CCR5, HPRT, AAVS1, Rosaor albumin gene.

In another aspect, provided herein are methods for providing afunctional protein lacking or deficient in a patient, for example fortreating genetic disorders. In certain embodiments, the methods compriseintegrating a sequence encoding the functional protein in a cell in asubject in need thereof using the ordered sequential administration ofnuclease(s) and transgene(s) as disclosed herein. In other embodiments,the methods comprise administering a genetically modified cell(expressing a functional version of one or more proteins aberrantlyexpressed in a subject) directly to the subject. Thus, an isolated cellmay be introduced into the subject (ex vivo cell therapy) or a cell maybe modified when it is part of the subject (in vivo). Also provided isthe use of the donors and/or nucleases described herein for thetreatment of a disorder, for example, in the preparation of medicamentfor treatment of a genetic disorder. In certain embodiments, theexogenous sequence is delivered using a viral vector, a non-viral vector(e.g., plasmid) and/or combinations thereof.

In any of the compositions and methods described, the nuclease(s) and/ortransgene(s) may be carried on an AAV vector, including but not limitedto AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9 and AAVrh10 or pseudotypedAAV such as AAV2/8, AAV8.2, AAV2/5 and AAV2/6 and the like. In certainembodiments, the nucleases and transgene donors are delivered using thesame AAV vector types. In other embodiments, the nucleases and transgenedonors are delivered using different AAV vector types. The nucleases andtransgenes may be delivered using two or more vectors, for example, twovectors where one carries the nuclease(s) (e.g., left and right ZFNs ofa ZFN pair, for example with a 2A peptide) and one carries thetransgene; or three vectors where one vector carries one nuclease of anuclease pair (e.g., left ZFN), a separate vector carries the othernuclease of a nuclease pair (e.g., right ZFN) and a third separatevector carries the transgene. In embodiments, in which two or morevectors or used, the vectors may be used at the same concentrations orin different ratios, for example, the nuclease vector(s) may beadministered at 2-fold, 3-fold, 4-fold, 5-fold or more higherconcentrations than the transgene vector(s).

In any of the compositions and methods described herein, the nuclease(s)may be delivered as mRNAs encoding said nucleases. In some embodiments,a single mRNA encoding the two nucleases of a nuclease pair wherein thecoding sequences for each nuclease are separated by a ‘self-cleaving’sequence (e.g., a 2A sequence) is described. In other embodiments, eachnuclease of a nuclease pair is comprised as a single mRNA, wherein twosingle mRNAs are used together to administer a nuclease pair. In any ofthe mRNA compositions described herein, the mRNA may be modified toincrease stability and/or efficiency of transcription, or may includemodified nucleosides (for examples, see U.S. Pat. Nos. 8,6919,66 and7,074,596), or the mRNA may be delivered via a formulated particle or asan encapsulated liposome (see, e.g. U.S. Pat. No. 5,976,567).

In any of the compositions and methods described herein, the transgenemay encode a protein, for example a functional version of a proteinlacking and/or aberrantly expressed in a disorder. In some embodiments,the transgene may encode a non-naturally occurring protein with enhancedcharacteristics as compared to its naturally occurring counterpart. Inany of the compositions or methods described herein, the transgene alsocomprises a transcriptional regulator while in others, it does not andtranscription is regulated by an endogenous regulator. In anotheraspect, the methods of the invention comprise a composition fortherapeutic treatment of a subject in need thereof. In some embodiments,the composition comprises engineered stem cells comprising a safe harborspecific nuclease, and a transgene donor. In other embodiments, thecomposition comprises engineered virus particles comprising transgenedonors and specific nucleases and/or modified mRNAs for performing invivo gene modification.

In any of the compositions or methods described herein, the cell may bea eukaryotic cell. Non-limiting examples of suitable cells includeeukaryotic cells or cell lines such as secretory cells (e.g., livercells, mucosal cells, salivary gland cells, pituitary cells, etc.),blood cells (red blood cells), red blood precursory cells, hepaticcells, muscle cells, stem cells (e.g., embryonic stem cells, inducedpluripotent stem cells, hepatic stem cells, hematopoietic stem cells(e.g., CD34+)) or endothelial cells (e.g., vascular, glomerular, andtubular endothelial cells). Thus, the target cells may be human cells,or cells of other mammals (including veterinary animals), especiallynonhuman primates (Macaca mulatta: rhesus macaque, Macaca fascicularis:cynomolous monkey) and mammals of the orders Rodenta (mice, rats,hamsters), Lagomorpha (rabbits), Carnivora (cats, dogs), andArteriodactyla (cows, pigs, sheep, goats, horses). In some aspects, thetarget cells comprise a tissue (e.g. liver). In some aspects, the targetcell is a stem cell (e.g., an embryonic stem cell, an inducedpluripotent stem cell, a hepatic stem cell, etc.) or animal embryo byany of the methods described herein, and then the embryo is implantedsuch that a live animal is born. The animal is then raised to sexualmaturity and allowed to produce offspring wherein at least some of theoffspring comprise the genomic modification. The cell can also comprisean embryo cell, for example, of a mouse, rat, rabbit or other mammaliancell embryo. The cell may be from any organism, for example human,non-human primate, mouse, rat, rabbit, cat, dog or other mammaliancells. The cell may be isolated or may be part of an organism (e.g.,subject).

In any of the methods and compositions described herein, the transgenemay be integrated into the endogenous safe harbor gene such that some,all or none of the endogenous gene is expressed, for example a fusionprotein with the integrated transgene. In some embodiments, theendogenous safe harbor gene is an albumin gene and the endogenoussequences are albumin sequences. The endogenous sequences may be presenton the amino (N)-terminal portion of the exogenous protein and/or on thecarboxy (C)-terminal portion of the exogenous protein. The albuminsequences may include full-length wild-type or mutant albumin sequencesor, alternatively, may include partial albumin amino acid sequences. Incertain embodiments, the albumin sequences (full-length or partial)serve to increase the serum half-life of the polypeptide expressed bythe transgene to which it is fused and/or as a carrier. In otherembodiments, the transgene comprises albumin sequences and is targetedfor insertion into another safe harbor within a genome. Furthermore, thetransgene may include an exogenous promoter (e.g., constitutive orinducible promoter) that drives its expression or its expression may bedriven by endogenous control sequences (e.g., endogenous albuminpromoter). In some embodiments, the donor includes additionalmodifications, including but not limited to codon optimization, additionof glycosylation sites, signal sequences and the like.

Furthermore, any of the methods described herein may further compriseadditional steps, including cold-shocking of the cells at any time (U.S.Pat. No. 8,772,008), partial hepatectomy or treatment with secondaryagents that enhance transduction and/or induce hepatic cells to undergocell cycling. Examples of secondary agents include gamma irradiation, UVirradiation, tritiated nucleotides such as thymidine, cis-platinum,etoposide, hydroxyurea, aphidicolin, prednisolone, carbon tetrachlorideand/or adenovirus.

The methods described herein can be practiced in vitro, ex vivo or invivo. In certain embodiments, the methods are performed in (and/orcompositions such as modified cells delivered to) a live, intact mammal.The mammal may be at any stage of development at the time of delivery,e.g., embryonic, fetal, neonatal, infantile, juvenile or adult.Additionally, targeted cells may be healthy or diseased. In certainembodiments, one or more of the compositions are delivered intravenously(e.g., to the liver via the hepatic portal vein, for example tail veininjection), intra-arterially, intraperitoneally, intramuscularly, intoliver parenchyma (e.g., via injection), into the hepatic artery (e.g.,via injection), and/or through the biliary tree (e.g., via injection).

For targeting the compositions to a particular type of cell, e.g.,platelets, fibroblasts, hepatocytes, hematopoietic stem/progenitor cellsetc., one or more of the administered compositions may be associatedwith a homing agent that binds specifically to a surface receptor of thecell. For example, the vector may be conjugated to a ligand (e.g.,galactose) for which certain hepatic system cells have receptors. Theconjugation may be covalent, e.g., a crosslinking agent such asglutaraldehyde, or noncovalent, e.g., the binding of an avidinatedligand to a biotinylated vector. Another form of covalent conjugation isprovided by engineering the AAV helper plasmid used to prepare thevector stock so that one or more of the encoded coat proteins is ahybrid of a native AAV coat protein and a peptide or protein ligand,such that the ligand is exposed on the surface of the viral particle.

A kit, comprising the compositions (e.g., genetically modified cells,ZFPs, CRISPR/Cas system and/or TALEs of the invention, is also provided.The kit may comprise nucleic acids encoding the nucleases, (e.g. RNAmolecules or nuclease-encoding genes contained in a suitable expressionvector or proteins), donor molecules, suitable host cell lines,instructions for performing the methods of the invention, and the like.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict schemes for varying the order of addition ofdonor and nuclease, depending on whether both are to be delivered inviral vectors or if the nuclease is delivered as mRNA. FIG. 1A shows themethod if the nuclease(s) is delivered as mRNA. In this case, the donorcontaining AAV virus is delivered to the cell, followed by delivery ofthe nuclease-encoding mRNA up to 48 hours after delivery of the donor.FIG. 1B shows the method if both donor and nuclease are delivered viaAAV viruses. In this instance, the nuclease-containing virus isdelivered first, taken up by the cells, and then the donor-containingvirus is delivered up to 48 hours after delivery of the nuclease virus.

FIG. 2 is a graph depicting transgene activity following insertion inprimary human hepatocytes. In this experiment, AAV2/6 virus particlescomprising a DNA donor encoding the secreted embryonic alkalinephosphatase (SEAP) reporter gene protein flanked by hALB specifichomology arms was introduced into the cells at a MOI of 1e5. mRNAsencoding human albumin specific-ZFNs were introduced to the cells atvarious time points from 0 to 72 hours following the introduction of thedonor containing AAV. Transgene expression was then measured 7 dayslater. The graph demonstrates that introduction of the nucleases 24-48hours after AAV-donor introduction lead to the optimum transgeneexpression.

FIGS. 3A to 3C depict the results of an investigation of varying theorder of addition of the nucleases and transgene donor for expression ofhuman Factor 9 (hF.IX). FIG. 3A is a schematic showing the insertion ofthe human F9 gene into the albumin locus in non-human primate (NHP)primary hepatocytes. The picture illustrates that the hF9 gene donor wasflanked by NHP (rhesus) albumin homology arms. FIG. 3B shows the initialtranslation product from the transgene insertion, and shows theproteolytic cleavage sites on the prepro hF.IX peptide. FIG. 3C showsthe expression of hF.IX detected in the cell supernatant. This graphshows the results using two NHP(rhesus) albumin specific ZFN pairs,36806/35396 and 37804/43043 where the mRNAs encoding the ZFNs were addedeither 24 or 48 hours following addition of the AAV-containing hF9transgene. The highest expression levels in this experiment wereobtained when the mRNAs encoding the ZFNs were added 24 hours after thetransgene containing AAV.

FIG. 4 is a Western blot depicting the expression of one of the two NHP(Rhesus) Albumin specific ZFN proteins following mRNA introduction inNHP (rhesus) primary hepatocytes. For detection of ZFNs an anti-FokIantibody was used while HSP90 served as loading control. The data showsthat ZFN protein levels peak approximately 8 hours post transfection andis nearly undetectable 48 hours post transfection.

FIGS. 5A and 5B depict nuclease-mediated targeted integration of atransgene in vitro in rhesus primary hepatocytes via sequentialadministration of nuclease and transgene where both the ZFNs and donorare delivered as viral vectors. Both the ZFN (specific for the rhesusalbumin gene) and hF9 donor were delivered via recombinant AAV2/6 virus,and the donor hF9 transgene was flanked with homology arms containingsequences homologous to those surrounding the cleavage site in therhesus albumin gene. The ZFN containing virus was added first, and thenthe donor comprising virus was added either on the same day or up to 48hours later. FIG. 5A depicts a graph showing the results of a hFIXspecific ELISA assay when the AAV particles were delivered at a ratio ofdonor AAV: ZFN1 AAV: ZFN2 AAV of 10:1:1, while FIG. 5B depicts a graphshowing the results when the ratios were varied (donor AAV: ZFN1 AAV:ZFN2 AAV of 3:1:1; 10:1:1 or 16:1:1). Both graphs show that the optimumhF.IX transgene expression was found when the donor-AAV transgene viruswas added 24 hours after the ZFN-AAV and that under these conditions alower Donor: ZFN ratio is beneficial.

FIG. 6 is a graph depicting hF.IX transgene expression when the hF9transgene was flanked with regions of homology to the human F9 gene,rather than homology arms that are homologous to the NHP albumin locuswhere the transgene was being inserted, a scheme that forces integrationof the donor only through NHEJ-dependent end capture. In this case,delay of the donor-AAV addition for 24 hours following ZFN-AAV treatmentwas still optimum for hF.IX expression.

FIGS. 7A and 7B depict the results of delayed hF9 transgene AAV-donorviral introduction where the hF9 transgene is flanked with homology armsfor the human albumin locus, not the NHP albumin locus, forcingintegration of the trangene through NHEJ-dependent end capture. FIG. 7Adepicts a characterization of the ZFN activity at days 4 and 8 posttransfection through an analysis of the percent of insertions anddeletions (“indels”) detected at the albumin cleavage site by the ZFNsunder the varying ZFN:donor virus ratio conditions. This data shows thatZFN activity decreases when higher amounts of donor virus are introducedon the same day. In contrast, when donor addition was delayed 24 hours,the negative impact the donor vector had on ZFN activity was no longerapparent even at a high donor:ZFN ratios. The data depicted in the day 8samples is from duplicate samples. FIG. 7B depicts the amount of hF9transgene expression in the same conditions as described in FIG. 7A.hF.IX expression is highest when the AAV-donor vector is introduced tothe culture 24 hours after the AAV-ZFN.

FIG. 8 depicts expression of an hF9 transgene in C57/B16 mice undervarious donor and ZFN virus delay protocols. The virus used was AAV2/8at a 6:1:1 ratio of donor-AAV:ZFN1-AAV: ZFN2-AAV virus. The conditionswere as follows: Donor added first, ZFN virus added 24 hours later(circles), administration of both virus at the same time (squares),administration of ZFN virus first, followed by donor virus 24 hourslater (triangles), administration of ZFN virus first, followed by donorvirus 72 hours later (inverted triangles), administration of ZFN virusfirst, followed by donor virus 120 hours later (diamonds). Controlgroups include ZFN only, Donor only and Vehicle only. P-values representstudent's T-test results between group 2 and 3 or groups 2 and 4,respectively.

FIGS. 9A and 9B depict ZFN activity and expression in the sameexperiment described above. FIG. 9A depicts the ZFN cleavage activity asmeasured by the percent of indels detected (as described above) at thealbumin locus in liver genomic DNA from all groups. FIG. 9B depicts ZFNexpression via Western blot analysis in the same liver tissues. ZFNactivity and expression was highest when the ZFN-containing AAV wasadded either alone or 3 days prior to the addition of thedonor-containing AAV.

FIG. 10 is a graph showing levels of hF.IX in mouse serum followinginfection with the AAV-ZFN and AAV-donor virus. AAV-donor was given tothe mice either one day before the AAV-ZFN virus, given the same day, orgiven 1 or 3 days after the AAV-ZFN. hF.IX was detected using ELISAfollowing serial bleeds of the different cohorts.

FIGS. 11A and 11B show hF.IX levels in treated animals. FIG. 11A is agraph depicting hF.IX levels in Rhesus plasma from 8 treated animals(Day 0 to Day 28) as determined by ELISA. AAV-donor was given to themonkeys either on the same day, or given 1 or 2 days after the AAV-ZFN.FIG. 11B shows the peak hF.IX levels from the same study achieved duringthe whole study duration.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods for integrating one moreexogenous donor sequences into a target site of a cell. The methods andcompositions involve sequential administration of (i) one or morenucleases that cleave the target sequence and (ii) one or more donorsequences that are integrated into the target sequence followingcleavage of the target sequence by the nuclease and in which there is adelay of minutes, hours or days between the administration of thenuclease(s) and the exogenous sequence(s). The methods and compositionsdescribed herein increase the efficiency of targeted gene correction orintegration of an exogenous sequence into an endogenous genomic locususing a targeted nuclease by adhering to specific methodologies ofsequential administration of the nuclease(s) and donor construct, wherethe order of the sequence is dependent upon the form used for deliveryof the nucleases and donor.

In particular, administration of the donor transgene (e.g., proteinencoding sequence or DNA sequence encoding a RNA such as an shRNA),after administration of the nuclease(s) when both nucleases and donorare supplied as viral particles results in increased transgeneintegration as compared to methods in which the donor and nucleases areadministered together (concurrently) or with a shorter or longer delay.The donor transgene may be administered from minutes to hours to daysafter the nuclease(s), for example, 8 to 72 hours (or any timetherebetween) or 4 days, 5 days, 6 days or even more. Alternatively,when the nuclease is administered as mRNA(s), it is preferred toadminister the viral particle comprising the donor transgene first,allow for sufficient time for uptake by the target cell, and then treatwith the nuclease encoding mRNAs. The nuclease encoding mRNAs may beadministered from minutes to hours to days after the donor(s), forexample, 8 to 72 hours (or any time therebetween) or 4 days, 5 days, 6days or even more. In either scenario, the cell is given sufficient timefor viral uptake. Without being bound by any particular theory, whenboth the nucleases and transgene donor are administered via virus, it ispossible that the two types of particles compete for the same uptakereceptors and diminish overall activity. Another possible mode ofcompetition between the viruses is after they enter the cell. Both ZFNand Donor virus have to first escape the endosomes in order to enter thenucleus. The resulting free single-stranded AAV genome then has to beconverted into a double-stranded form to a) either serve as Donor or b)to be transcribed and produced the ZFN protein. Any of these steps couldbe rate-limiting and could therefore be sensitive towards AAVcompetition. It is also beneficial that if sufficient time between theadministration of the two particle types is allowed, the nuclease viruswill have been taken up and the nucleases will have started acting tocreate the specific DSB at the endogenous genomic target before thetransgene donor virus is introduced. When the nuclease is supplied asmRNA, competition for uptake receptors is not an issue as the mRNA istaken up immediately during the transfection procedure (see FIG. 1). Insome specific cell types (e.g. CD34+ hematopoietic stem cells, it may bepreferable to treat the cells with the donor-AAV immediately followingnuclease introduction via mRNA.

An exogenous sequence can encode any protein or peptide involved inhemophilia, for example F8, F.IX and/or functional fragments thereof.Also disclosed are methods of treating a hemophilia using a cell asdescribed herein and/or by modifying a cell (ex vivo or in vivo) asdescribed herein.

The transgene may encode a protein product, for example a functionalversion of a protein that is lacking, aberrantly expressed and/ornon-functional in the cell, for example a protein lacking in a subjectwith hemophilia (e.g., Factor VII, F8, F.IX, Factor X, and/or functionalfragments thereof), a protein lacking in a subject with a lysosomalstorage disease, a protein lacking in a subject with a hemoglobinopathy,and/or a protein lacking in a subject with a metabolic disorder. See,e.g., U.S. Publication Nos. 20120128635; 20140093913; 20140080216 and20140155468.

The genomically-modified cells described herein are typically modifiedvia nuclease-mediated (ZFN, TALEN and/or CRISPR/Cas) targetedintegration to insert a sequence encoding a therapeutic protein into thegenome of one or more cells of the subject (in vivo or ex vivo), suchthat the cells produce the protein in vivo.

In certain embodiments, the methods further comprise inducing cells ofthe subject, particularly liver cells, to proliferate (enter the cellcycle), for example, by partial hepatectomy and/or by administration ofone or more compounds that induce hepatic cells to undergo cell cycling.Subjects include but are not limited to humans, non-human primates,veterinary animals such as cats, dogs, rabbits, rats, mice, guinea pigs,cows, pigs, horses, goats and the like.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein. See, e.g.,U.S. Pat. No. 8,586,526.

Zinc finger and TALE binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger or TALE protein. Therefore, engineered DNA bindingproteins (zinc fingers or TALEs) are proteins that are non-naturallyoccurring. Non-limiting examples of methods for engineering DNA-bindingproteins are design and selection. A designed DNA binding protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPand/or TALE designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; 6,534,261; and 8,586,526 see also WO 98/53058; WO98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; WO95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO01/60970 WO 01/88197 and WO 02/099084.

“TtAgo” is a prokaryotic Argonaute protein thought to be involved ingene silencing. TtAgo is derived from the bacteria Thermus thermophilus.See, e.g., Swarts et al, (2014) Nature 507: 258-261; G. Sheng et al.,(2013) Proc. Natl. Acad. Sci. U.S.A. 111, 652). A “TtAgo system” is allthe components required including, for example, guide DNAs for cleavageby a TtAgo enzyme. “Recombination” refers to a process of exchange ofgenetic information between two polynucleotides, including but notlimited to, donor capture by non-homologous end joining (NHEJ) andhomologous recombination. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells via homology-directed repair mechanisms. This processrequires nucleotide sequence homology, uses a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target. Withoutwishing to be bound by any particular theory, such transfer can involvemismatch correction of heteroduplex DNA that forms between the brokentarget and the donor, and/or “synthesis-dependent strand annealing,” inwhich the donor is used to resynthesize genetic information that willbecome part of the target, and/or related processes. Such specialized HRoften results in an alteration of the sequence of the target moleculesuch that part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break, can be introduced into the cell. The presence of thedouble-stranded break has been shown to facilitate integration of thedonor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingernucleases and/or TALENs can be used for additional double-strandedcleavage of additional target sites within the cell.

In certain embodiments of methods for targeted recombination and/orreplacement and/or alteration of a sequence in a region of interest incellular chromatin, a chromosomal sequence is altered by homologousrecombination with an exogenous “donor” nucleotide sequence. Suchhomologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present.

In any of the methods described herein, the first nucleotide sequence(the “donor sequence”) can contain sequences that are homologous, butnot identical, to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to 99% (or any integer therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 100 contiguous base pairs. Incertain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50-1,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 1,000, that are homologous oridentical to sequences in the region of interest. In other embodiments,the donor sequence is non-homologous to the first sequence, and isinserted into the genome by non-homologous recombination mechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or noncoding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid (donor) sequence may produce one or more RNAmolecules (e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis),microRNAs (miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 20070218528 and 2008/0131962,incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogeneous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more nuclease domains ortranscriptional regulatory domains such as activation or repressiondomains) and fusion nucleic acids (for example, a nucleic acid encodingthe fusion protein described supra). Examples of the second type offusion molecule include, but are not limited to, a fusion between atriplex-forming nucleic acid and a polypeptide, and a fusion between aminor groove binder and a nucleic acid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP as described herein.Thus, gene inactivation may be partial or complete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a DNA-bindingdomain (ZFP, TALE) is fused to a cleavage domain (e.g., endonucleasedomain such as FokI, meganuclease domain, etc.), the DNA-binding domainand the cleavage domain are in operative linkage if, in the fusionpolypeptide, the DNA-binding domain portion is able to bind its targetsite and/or its binding site, while the cleavage (nuclease) domain isable to cleave DNA in the vicinity of the target site. The nucleasedomain may also exhibit DNA-binding capability (e.g., a nuclease fusedto a ZFP or TALE domain that also can bind to DNA). Similarly, withrespect to a fusion polypeptide in which a DNA-binding domain is fusedto an activation or repression domain, the DNA-binding domain and theactivation or repression domain are in operative linkage if, in thefusion polypeptide, the DNA-binding domain portion is able to bind itstarget site and/or its binding site, while the activation domain is ableto upregulate gene expression or the repression domain is able todownregulate gene expression.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “safe harbor” locus is a locus within the genome wherein a gene may beinserted without any deleterious effects on the host cell. Mostbeneficial is a safe harbor locus in which expression of the insertedgene sequence is not perturbed by any read-through expression fromneighboring genes. Non-limiting examples of safe harbor loci that aretargeted by nuclease(s) include CCR5, CCR5, HPRT, AAVS1, Rosa andalbumin. See, e.g., U.S. Pat. Nos. 7,888,121; 7,972,854; 7,914,796;7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S. Patent Publications20030232410; 20050208489; 20050026157; 20060063231; 20080159996;201000218264; 20120017290; 20110265198; 20130137104; 20130122591;20130177983; 20130177960; and 20130122591 and U.S. Pat. No. 9,873,894).

Nucleases

Described herein are compositions, particularly nucleases, that areuseful in integration of a donor sequence in the genome of a cell fromor in a subject. In certain embodiments, the nuclease is naturallyoccurring. In other embodiments, the nuclease is non-naturallyoccurring, i.e., engineered in the DNA-binding domain and/or cleavagedomain. For example, the DNA-binding domain of a naturally-occurringnuclease may be altered to bind to a selected target site (e.g., ameganuclease that has been engineered to bind to site different than thecognate binding site). In other embodiments, the nuclease comprisesheterologous DNA-binding and cleavage domains (e.g., zinc fingernucleases; TAL-effector domain DNA binding proteins; meganucleaseDNA-binding domains with heterologous cleavage domains, or megaTALs:fusions between a TALE DNA binding protein and a homing endonuclease ormeganuclease) and/or a Ttago or CRISPR/Cas system utilizing anengineered single guide RNA).

A. DNA-Bbinding Domains

Any DNA-binding domain can be used in the compositions and methodsdisclosed herein, including but not limited to a zinc finger DNA-bindingdomain, a TALE DNA binding domain, the DNA-binding portion of aCRISPR/Cas nuclease, a Ttago nuclease, or a DNA-binding domain from ameganuclease.

The DNA-binding domain can be bind to any target sequence. In certainembodiments, the DNA-binding domain binds to an endogenous sequence, forexample a safe harbor within the genome. Non-limiting examples of safeharbor loci that can be targeted by the DNA-binding domain of one ormore nuclease(s) include CCR5, CCR5, HPRT, AAVS1, Rosa and albumin. See,e.g., U.S. Pat. Nos. 7,888,121; 7,972,854; 7,914,796; 7,951,925;8,110,379; 8,409,861; 8,586,526; U.S. Patent Publications 20030232410;20050208489; 20050026157; 20060063231; 20080159996; 201000218264;20120017290; 20110265198; 20130137104; 20130122591; 20130177983,20130177960 and U.S. Pat. No. 9,873,894).

In certain embodiments, the nuclease is a naturally occurring orengineered (non-naturally occurring) meganuclease (homing endonuclease).Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce,I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI,I-TevII and I-TevIII. Their recognition sequences are known. See alsoU.S. Pat. Nos. 5,420,032; 6,833,252; Belfort et al. (1997) Nucleic AcidsRes. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al.(1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet.12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast etal. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabscatalogue. Engineered meganucleases are described for example in U.S.Patent Publication No. 20070117128. The DNA-binding domains of thehoming endonucleases and meganucleases may be altered in the context ofthe nuclease as a whole (i.e., such that the nuclease includes thecognate cleavage domain) or may be fused to a heterologous cleavagedomain. DNA-binding domains from meganucleases may also exhibit nucleaseactivity.

In other embodiments, the DNA-binding domain comprises a naturallyoccurring or engineered (non-naturally occurring) TAL effector DNAbinding domain. See, e.g., U.S. Pat. No. 8,586,526, incorporated byreference in its entirety herein. The plant pathogenic bacteria of thegenus Xanthomonas are known to cause many diseases in important cropplants. Pathogenicity of Xanthomonas depends on a conserved type IIIsecretion (T3S) system which injects more than 25 different effectorproteins into the plant cell. Among these injected proteins aretranscription activator-like (TAL) effectors which mimic planttranscriptional activators and manipulate the plant transcriptome (seeKay et al (2007) Science 318:648-651). These proteins contain a DNAbinding domain and a transcriptional activation domain. One of the mostwell characterized TAL-effectors is AvrBs3 from Xanthomonas campestgrispv. Vesicatoria (see Bonas et al (1989) Mol Gen Genet 218: 127-136 andWO2010079430). TAL-effectors contain a centralized domain of tandemrepeats, each repeat containing approximately 34 amino acids, which arekey to the DNA binding specificity of these proteins. In addition, theycontain a nuclear localization sequence and an acidic transcriptionalactivation domain (for a review see Schornack S, et al (2006) J PlantPhysiol 163(3): 256-272). In addition, in the phytopathogenic bacteriaRalstonia solanacearum two genes, designated brg11 and hpx17 have beenfound that are homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GM11000 and in the biovar 4 strain RS1000(See Heuer et al (2007) Appl and Envir Micro 73(13): 4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas. See, e.g., U.S. See, e.g., U.S. Pat. No.8,586,526, incorporated by reference in its entirety herein.

Specificity of these TAL effectors depends on the sequences found in thetandem repeats. The repeated sequence comprises approximately 102 bp andthe repeats are typically 91-100% homologous with each other (Bonas etal, ibid). Polymorphism of the repeats is usually located at positions12 and 13 and there appears to be a one-to-one correspondence betweenthe identity of the hypervariable diresidues (the repeat variablediresidue or RVD region) at positions 12 and 13 with the identity of thecontiguous nucleotides in the TAL-effector's target sequence (see Moscouand Bogdanove, (2009) Science 326:1501 and Boch et al (2009) Science326:1509-1512). Experimentally, the natural code for DNA recognition ofthese TAL-effectors has been determined such that an HD sequence atpositions 12 and 13 (Repeat Variable Diresidue or RVD) leads to abinding to cytosine (C), NG binds to T, NI to A, C, G or T, NN binds toA or G, and ING binds to T. These DNA binding repeats have beenassembled into proteins with new combinations and numbers of repeats, tomake artificial transcription factors that are able to interact with newsequences and activate the expression of a non-endogenous reporter genein plant cells (Boch et al, ibia).

Engineered TAL proteins have been linked to a FokI cleavage half domainto yield a TAL effector domain nuclease fusion (TALEN), including TALENswith atypical RVDs. See, e.g., U.S. Pat. No. 8,586,526. Thus, in someembodiments, the TALENs comprise a TAL effector DNA-binding domain and arestriction endonuclease domain (e.g., FokI).

In some instances, TAL DNA binding domains have been linked to homingendonucleases/meganucleases to make “MegaTALs”. These fusion proteinsrely on the low cutting frequency of meganucleases naturally in anattempt to reduce any off-site cleavage by an engineered nuclease whileexploiting the TAL DNA binding domain to direct the site specificcleavage (see Boissel (2013) Nucl Acid Res 1-11).

In still further embodiments, the TALEN comprises a compact TALEN. Theseare single chain fusion proteins linking a TALE DNA binding domain to aTevl nuclease domain. The fusion protein can act as either a nickaselocalized by the TALE region, or can create a double strand break,depending upon where the TALE DNA binding domain is located with respectto the Tevl nuclease domain (see Beurdeley et al (2013) Nat Comm: 1-8DOI: 10.1038/ncomms2782).

In addition, the nuclease domain of TALENs as described herein may alsoexhibit DNA-binding functionality and any TALENs may be used incombination with additional TALENs (e.g., one or more TALENs (cTALENsand/or FokI-TALENs) with one or more mega-TALEs.

In certain embodiments, the DNA binding domain comprises a zinc fingerprotein. Preferably, the zinc finger protein is non-naturally occurringin that it is engineered to bind to a target site of choice. See, forexample, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al.(2001) Ann. Rev. Biochem. 70:313 -340; Isalan et al. (2001) NatureBiotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol.12:632 -637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416;U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558;7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635;7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528;2005/0267061, all incorporated herein by reference in their entireties.

An engineered zinc finger binding domain can have a novel bindingspecificity, compared to a naturally-occurring zinc finger protein.Engineering methods include, but are not limited to, rational design andvarious types of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

In addition, as disclosed in these and other references, DNA-bindingdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

In some embodiments, the TALEN comprises a endonuclease (e.g., FokI)cleavage domain or cleavage half-domain. In other embodiments, theTALE-nuclease is a mega TAL. These mega TAL nucleases are fusionproteins comprising a TALE DNA binding domain and a meganucleasecleavage domain. The meganuclease cleavage domain is active as a monomerand does not require dimerization for activity. (See Boissel et al.,(2013) Nucl Acid Res: 1-13, doi: 10.1093/nar/gkt1224).

In still further embodiments, the nuclease comprises a compact TALEN.These are single chain fusion proteins linking a TALE DNA binding domainto a TevI nuclease domain. The fusion protein can act as either anickase localized by the TALE region, or can create a double strandbreak, depending upon where the TALE DNA binding domain is located withrespect to the TevI nuclease domain (see Beurdeley et al (2013) NatComm: 1-8 DOI: 10.1038/ncomms2782). In addition, the nuclease domain mayalso exhibit DNA-binding functionality. Any TALENs may be used incombination with additional TALENs (e.g., one or more TALENs (cTALENs orFokI-TALENs) with one or more mega-TALEs.

Selection of target sites and methods for design and construction offusion proteins (and polynucleotides encoding same) are known to thoseof skill in the art and described in detail in U.S. Pat. Nos. 6,140,081;5,789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453;6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO 98/53058; WO98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

In some embodiments, the DNA binding domain is part of a TtAgo system(see Swarts et al, ibid; Sheng et al, ibid). In eukaryotes, genesilencing is mediated by the Argonaute (Ago) family of proteins. In thisparadigm, Ago is bound to small (19-31 nt) RNAs. This protein-RNAsilencing complex recognizes target RNAs via Watson-Crick base pairingbetween the small RNA and the target and endonucleolytically cleaves thetarget RNA (Vogel (2014) Science 344:972-973). In contrast, prokaryoticAgo proteins bind to small single-stranded DNA fragments and likelyfunction to detect and remove foreign (often viral) DNA (Yuan et al.,(2005) Mol. Cell 19, 405; Olovnikov, et al. (2013) Mol. Cell 51, 594;Swarts et al., ibid). Exemplary prokaryotic Ago proteins include thosefrom Aquifex aeolicus, Rhodobacter sphaeroides, and Thermusthermophilus.

One of the most well-characterized prokaryotic Ago protein is the onefrom T. thermophilus (TtAgo; Swarts et al. ibid). TtAgo associates witheither 15 nt or 13-25 nt single-stranded DNA fragments with 5′ phosphategroups. This “guide DNA” bound by TtAgo serves to direct the protein-DNAcomplex to bind a Watson-Crick complementary DNA sequence in athird-party molecule of DNA. Once the sequence information in theseguide DNAs has allowed identification of the target DNA, the TtAgo-guideDNA complex cleaves the target DNA. Such a mechanism is also supportedby the structure of the TtAgo-guide DNA complex while bound to itstarget DNA (G. Sheng et al., ibid). Ago from Rhodobacter sphaeroides(RsAgo) has similar properties (Olivnikov et al. ibid).

Exogenous guide DNAs of arbitrary DNA sequence can be loaded onto theTtAgo protein (Swarts et al. ibid.). Since the specificity of TtAgocleavage is directed by the guide DNA, a TtAgo-DNA complex formed withan exogenous, investigator-specified guide DNA will therefore directTtAgo target DNA cleavage to a complementary investigator-specifiedtarget DNA. In this way, one may create a targeted double-strand breakin DNA. Use of the TtAgo-guide DNA system (or orthologous Ago-guide DNAsystems from other organisms) allows for targeted cleavage of genomicDNA within cells. Such cleavage can be either single- ordouble-stranded. For cleavage of mammalian genomic DNA, it would bepreferable to use of a version of TtAgo codon optimized for expressionin mammalian cells. Further, it might be preferable to treat cells witha TtAgo-DNA complex formed in vitro where the TtAgo protein is fused toa cell-penetrating peptide. Further, it might be preferable to use aversion of the TtAgo protein that has been altered via mutagenesis tohave enhanced activity at 37 degrees Celsius. Ago-RNA-mediated DNAcleavage could be used to affect a panopoly of outcomes including geneknock-out, targeted gene addition, gene correction, targeted genedeletion using techniques standard in the art for exploitation of DNAbreaks.

In certain embodiments, the nuclease comprises a CRISPR/Cas system. TheCRISPR (clustered regularly interspaced short palindromic repeats)locus, which encodes RNA components of the system, and the cas(CRISPR-associated) locus, which encodes proteins (Jansen et al., 2002.Mol. Microbiol. 43: 1565-1575; Makarova et al., 2002. Nucleic Acids Res.30: 482-496; Makarova et al., 2006. Biol. Direct 1: 7; Haft et al.,2005. PLoS Comput. Biol. 1: e60) make up the gene sequences of theCRISPR/Cas nuclease system. CRISPR loci in microbial hosts contain acombination of CRISPR-associated (Cas) genes as well as non-coding RNAelements capable of programming the specificity of the CRISPR-mediatednucleic acid cleavage.

The Type II CRISPR is one of the most well characterized systems andcarries out targeted DNA double-strand break in four sequential steps.First, two non-coding RNA, the pre-crRNA array and tracrRNA, aretranscribed from the CRISPR locus. Second, tracrRNA hybridizes to therepeat regions of the pre-crRNA and mediates the processing of pre-crRNAinto mature crRNAs containing individual spacer sequences. Third, themature crRNA:tracrRNA complex directs Cas9 to the target DNA viaWatson-Crick base-pairing between the spacer on the crRNA and theprotospacer on the target DNA next to the protospacer adjacent motif(PAM), an additional requirement for target recognition. Finally, Cas9mediates cleavage of target DNA to create a double-stranded break withinthe protospacer. Activity of the CRISPR/Cas system comprises of threesteps: (i) insertion of alien DNA sequences into the CRISPR array toprevent future attacks, in a process called ‘adaptation’, (ii)expression of the relevant proteins, as well as expression andprocessing of the array, followed by (iii) RNA-mediated interferencewith the alien nucleic acid. Thus, in the bacterial cell, several of theso-called ‘Cas’ proteins are involved with the natural function of theCRISPR/Cas system and serve roles in functions such as insertion of thealien DNA etc.

In certain embodiments, Cas protein may be a “functional derivative” ofa naturally occurring Cas protein. A “functional derivative” of a nativesequence polypeptide is a compound having a qualitative biologicalproperty in common with a native sequence polypeptide. “Functionalderivatives” include, but are not limited to, fragments of a nativesequence and derivatives of a native sequence polypeptide and itsfragments, provided that they have a biological activity in common witha corresponding native sequence polypeptide. A biological activitycontemplated herein is the ability of the functional derivative tohydrolyze a DNA substrate into fragments. The term “derivative”encompasses both amino acid sequence variants of polypeptide, covalentmodifications, and fusions thereof. Suitable derivatives of a Caspolypeptide or a fragment thereof include but are not limited tomutants, fusions, covalent modifications of Cas protein or a fragmentthereof. Cas protein, which includes Cas protein or a fragment thereof,as well as derivatives of Cas protein or a fragment thereof, may beobtainable from a cell or synthesized chemically or by a combination ofthese two procedures. The cell may be a cell that naturally produces Casprotein, or a cell that naturally produces Cas protein and isgenetically engineered to produce the endogenous Cas protein at a higherexpression level or to produce a Cas protein from an exogenouslyintroduced nucleic acid, which nucleic acid encodes a Cas that is sameor different from the endogenous Cas. In some case, the cell does notnaturally produce Cas protein and is genetically engineered to produce aCas protein.

Exemplary CRISPR/Cas nuclease systems targeted to safe harbor and othergenes are disclosed for example, in U.S. Pat. No. 9,873,894.

Thus, the nuclease can comprise any DNA-binding domain (e.g., zincfinger protein, TALE, single guide RNA) that specifically binds to atarget site in any gene.

B. Cleavage Domains

Any suitable cleavage domain can be operatively linked to a DNA-bindingdomain to form a nuclease, such as a zinc finger nuclease, a TALEN, or aCRISPR/Cas nuclease system.

As noted above, the cleavage domain may be heterologous to theDNA-binding domain, for example a zinc finger DNA-binding domain and acleavage domain from a nuclease or a TALEN DNA-binding domain and acleavage domain, or meganuclease DNA-binding domain and cleavage domainfrom a different nuclease. Heterologous cleavage domains can be obtainedfrom any endonuclease or exonuclease. Exemplary endonucleases from whicha cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any number of nucleotides or nucleotide pairs canintervene between two target sites (e.g., from 2 to 50 nucleotide pairsor more). In general, the site of cleavage lies between the targetsites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31, 978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-Fok Ifusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474; 20060188987;20070305346 and 20080131962, the disclosures of all of which areincorporated by reference in their entireties herein. Amino acidresidues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496,498, 499, 500, 531, 534, 537, and 538 of Fok I are all targets forinfluencing dimerization of the Fok I cleavage half-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Pat. No. 7,914,796, the disclosure of which is incorporated by referencein its entirety for all purposes.

In certain embodiments, the engineered cleavage half-domain comprisesmutations at positions 486, 499 and 496 (numbered relative to wild-typeFokI), for instance mutations that replace the wild type Gln (Q) residueat position 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See US Pat. No. 8,623,618, incorporated byreference herein)). In other embodiments, the engineered cleavage halfdomain comprises the “Sharkey” and/or “Sharkey' ” mutations (see Guo etal, (2010) J. Mol. Biol. 400(1):96-107).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. Pat. Nos.7,888,121; 7,914,796; 8,034,598 and 8,823,618.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in WO 2009/042163 and20090068164. Nuclease expression constructs can be readily designedusing methods known in the art. See, e.g., U.S. Pat. Nos. 7,888,121;8,409,861; 7,972,854; 7,914,796; 7,951,925; 7,919,313; and U.S. PatentPublications 20030232410; 20050208489; 20050026157; 20050064474;20060188987; 20060063231; and International Publication WO 07/014275.Expression of the nuclease may be under the control of a constitutivepromoter or an inducible promoter, for example the galactokinasepromoter which is activated (de-repressed) in the presence of raffinoseand/or galactose and repressed in presence of glucose.

Thus, the nuclease specifically targets any site into which it isdesired to insert a donor (transgene).

Target Sites

As described in detail above, DNA-binding domains can be engineered tobind to any sequence of choice, for example in a safe-harbor locus suchas CCRS, HPRT, albumin, Rosa, CXCR4 and AAVS1. An engineered DNA-bindingdomain can have a novel binding specificity, compared to anaturally-occurring DNA-binding domain. Engineering methods include, butare not limited to, rational design and various types of selection.Rational design includes, for example, using databases comprisingtriplet (or quadruplet) nucleotide sequences and individual zinc fingeramino acid sequences, in which each triplet or quadruplet nucleotidesequence is associated with one or more amino acid sequences of zincfingers which bind the particular triplet or quadruplet sequence. See,for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261,incorporated by reference herein in their entireties. Rational design ofTAL-effector domains can also be performed. See, e.g., U.S. Pat. No.8,586,526.

Exemplary selection methods applicable to DNA-binding domains, includingphage display and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466;6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO00/27878; WO 01/88197 and GB 2,338,237. In addition, enhancement ofbinding specificity for zinc finger binding domains has been described,for example, in co-owned WO 02/077227.

Selection of target sites; nucleases and methods for design andconstruction of fusion proteins (and polynucleotides encoding same) areknown to those of skill in the art and described in detail in U.S.Patent Publication Nos. 20050064474 and 20060188987, incorporated byreference in their entireties herein.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-fingered zinc finger proteins) may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids. See, e.g., U.S. Pat. Nos. 6,479,626;6,903,185; and 7,153,949 for exemplary linker sequences 6 or more aminoacids in length. The proteins described herein may include anycombination of suitable linkers between the individual DNA-bindingdomains of the protein. See, also, U.S. Pat. No. 8,586,526.

For treatment of a disorder (e.g., hemophilia, a lysosomal storagedisorder, a metabolic disorder, a hemoglobinopathy) via targetedinsertion of a sequence encoding a functional version of one or moreproteins aberrantly expressed in a subject with the disorder, anydesired site of insertion in the genome of the subject is cleaved with anuclease, which stimulates targeted insertion of the donorpolynucleotide carrying the protein-encoding sequence. DNA-bindingdomains of the nucleases may be targeted to any desired site in thegenome. In certain embodiments, the DNA-binding domain of the nucleaseis targeted to an endogenous safe harbor locus, for example anendogenous albumin locus.

Donor Sequences

Any donor sequence can be integrated using the methods described herein,including one or more DNA sequences. For treating a disorder in which aprotein is aberrantly expressed (lacking and/or non-functional), thedonor sequence (also called an “exogenous sequence” or “donor” or“transgene”) comprises a sequence encoding a functional version of theprotein, or part thereof, to result in a sequence encoding andexpressing a functional protein following donor integration.Non-limiting examples of suitable proteins include clotting factorprotein transgenes for treatment of hemophilias, for example, Factor VII(F7), Factor VIII (F8), Factor IX (F9 or F.IX or FIX) and/or Factor X(F10 or FX), including functional fragments of these proteins. Incertain embodiments, the B-domain of the F8 protein is deleted. See,e.g., Chuah et al. (2003) Blood 101(5):1734-1743. In other embodiments,the transgene comprises a sequence encoding a functional F.IX protein,or part thereof, to result in a sequence encoding and expressing afunction F.IX protein following donor integration. See, also, U.S. Pat.No. 10,081,661.

The donor molecule may be inserted into an endogenous gene such thatall, some or none of the endogenous gene is expressed. For example, atransgene comprising functional protein sequences as described hereinmay be inserted into an endogenous albumin locus such that some or noneof the endogenous albumin is expressed with the transgene.

The donor (transgene) sequence is introduced into the cell sequentially(e.g., prior to, or subsequent to), expression of the fusion protein(s)(e.g., nucleases). The donor polynucleotide may contain sufficienthomology (continuous or discontinuous regions) to a genomic sequence tosupport homologous recombination (or homology-directed repair) betweenit and the genomic sequence to which it bears homology or,alternatively, donor sequences can be integrated via non-HDR mechanisms(e.g., NHEJ donor capture), in which case the donor polynucleotide(e.g., vector) need not containing sequences that are homologous to theregion of interest in cellular chromatin. See, e.g., U.S. Pat. Nos.7,888,121 and 7,972,843 and 8,703,489 and U.S. Publication Nos.20110281361 and 20110207221.

A donor sequence may also be used for gene correction or alteration ofan endogenous gene. Such a donor may be an oligonucleotide used forcorrection of a mutation in an endogenous gene or may be used to alterthe wild type sequence to impart an improvement in gene productcharacteristics. The donor may also be used to correct or altersequences in coding sequences, regulatory sequences or other non-codingsequences.

The donor polynucleotide can be DNA or RNA, single-stranded,double-stranded or partially single- and partially double-stranded andcan be introduced into a cell in linear or circular (e.g., minicircle)form. See, e.g., U.S. Pat. No. 8,703,489 and U.S. Publication Nos.20110281361 and 20110207221. If introduced in linear form, the ends ofthe donor sequence can be protected (e.g., from exonucleolyticdegradation) by methods known to those of skill in the art. For example,one or more dideoxynucleotide residues are added to the 3′ terminus of alinear molecule and/or self-complementary oligonucleotides are ligatedto one or both ends. See, for example, Chang et al. (1987) Proc. Natl.Acad. Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889.Additional methods for protecting exogenous polynucleotides fromdegradation include, but are not limited to, addition of terminal aminogroup(s) and the use of modified internucleotide linkages such as, forexample, phosphorothioates, phosphoramidates, and O-methyl ribose ordeoxyribose residues. A polynucleotide can be introduced into a cell aspart of a vector molecule having additional sequences such as, forexample, replication origins, promoters and genes encoding antibioticresistance. Moreover, donor polynucleotides can be introduced as nakednucleic acid, as nucleic acid complexed with an agent such as a liposomeor poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV,herpesvirus, retrovirus, lentivirus).

The donor is generally inserted so that its expression is driven by theendogenous promoter at the integration site (e.g., the endogenousalbumin promoter when the donor is integrated into the patient's albuminlocus). Thus, the transgene typically lacks control elements (e.g.,promoter and/or enhancer) that drive its expression (e.g., also referredto as a “promoterless construct”). Nonetheless, it will be apparent thatthe donor may comprise a promoter and/or enhancer, for example aconstitutive promoter or an inducible or tissue specific (e.g., liver-or platelet-specific) promoter that drives expression of the functionalprotein upon integration.

The donor sequence can be integrated specifically into any target siteof choice, thereby eliminating the issues associated with randomintegration in traditional gene therapy.

When endogenous (e.g., albumin) sequences are expressed with thetransgene, the endogenous sequences may be full-length sequences(wild-type or mutant) or partial sequences. Preferably the albuminsequences are functional. In certain embodiments, the endogenoussequences are albumin sequences that may be expressed with the transgene(either from the endogenous locus or as part of the transgene).Non-limiting examples of the function of these full length or partialalbumin sequences include increasing the serum half-life of thepolypeptide expressed by the transgene (e.g., therapeutic gene) and/oracting as a carrier.

Furthermore, although not required for expression, exogenous sequencesmay also include transcriptional or translational regulatory sequences,for example, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

Any of the donor sequences may include one or more of the followingmodifications: codon optimization (e.g., to human codons) and/oraddition of one or more glycosylation sites. See, e.g., McIntosh et al.(2013) Blood (17):3335-44.

Delivery

The nucleases, polynucleotides encoding these nucleases, donorpolynucleotides and compositions comprising the proteins and/orpolynucleotides described herein may be delivered in vivo or ex vivo byany suitable means.

Methods of delivering nucleases as described herein are described, forexample, in U.S. Pat. Nos. 8,586,526; 6,453,242; 6,503,717; 6,534,261;6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539;7,013,219; and 7,163,824 the disclosures of all of which areincorporated by reference herein in their entireties.

Nucleases and/or donor constructs as described herein may also bedelivered using vectors containing sequences encoding one or more of thezinc finger protein(s), TALEN protein(s) and/or a CRISPR/Cas system. Anyvector systems may be used including, but not limited to, plasmidvectors, retroviral vectors, lentiviral vectors, adenovirus vectors,poxvirus vectors; herpesvirus vectors and adeno-associated virusvectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882; 6,824,978;6,933,113; 6,979,539; 7,013,219; and 7,163,824, incorporated byreference herein in their entireties. Furthermore, it will be apparentthat any of these vectors may comprise one or more of the sequencesneeded. Thus, when one or more nucleases and a donor construct areintroduced into the cell, the nucleases and/or donor polynucleotide maybe carried on the same vector or on different vectors. When multiplevectors are used, each vector may comprise a sequence encoding one ormultiple nucleases and/or donor constructs. In certain embodiments, onevector is used to carry both the transgene and nuclease(s). In otherembodiments, two vector are used (the same or different vector types),where one vector carries the nuclease(s) (e.g., left and right ZFNs of aZFN pair, for example with a 2A peptide) and one carries the transgene.In still further embodiments, three vectors are used where the firstvector carries one nuclease of a nuclease pair (e.g., left ZFN), thesecond vector carries the other nuclease of a nuclease pair (e.g., rightZFN) and the third vector carries the transgene.

The donors and/or nuclease may be used at any suitable concentrations.In certain embodiments, the donor and separate nuclease vector(s) areused the same concentration. In other embodiments, the donor andseparate nuclease vector(s) are used at different concentrations, forexample, 2-, 3-, 4-, 5-, 10- or more fold of one vector than other(e.g., more donor vector(s) than nuclease vector(s). When AAV vectorsare used for delivery, for example, the donor-and/or nuclease-comprisingviral vector(s) are between 1×10⁸ and 1×10¹³ particles per dose (e.g.,cell or animal).

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding nucleases and donor constructs incells (e.g., mammalian cells) and target tissues. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome or poloxamer.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of in vivo delivery of engineered DNA-binding proteins and fusionproteins comprising these binding proteins, see, e.g., Rebar (2004)Expert Opinion Invest. Drugs 13(7):829-839; Rossi et al. (2007) NatureBiotech. 25(12):1444-1454 as well as general gene delivery referencessuch as Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology Doerfler and Bohm (eds.) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that areuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., CancerRes.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGenelC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet al (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered nucleases and/or donors take advantage ofhighly evolved processes for targeting a virus to specific cells in thebody and trafficking the viral payload to the nucleus. Viral vectors canbe administered directly to patients (in vivo) or they can be used totreat cells in vitro and the modified cells are administered to patients(ex vivo). Conventional viral based systems for the delivery ofnucleases and/or donors include, but are not limited to, retroviral,lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplexvirus vectors for gene transfer. Integration in the host genome ispossible with the retrovirus, lentivirus, and adeno-associated virusgene transfer methods, often resulting in long term expression of theinserted transgene. Additionally, high transduction efficiencies havebeen observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors is described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellen et al., Immunol Immunother 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748 -55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4,AAV5, AAV6, AAV8, AAV9 and AAVrh10 or pseudotyped AAV such as AAV2/8,AAV8.2, AAV2/5 and AAV2/6 and any novel AAV serotype can also be used inaccordance with the present invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:15-10 (1996); Sterman et al., Hum. Gene Ther. 9:7 1083-1089(1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al.,Hum. Gene Ther. 5:597 -613 (1997); Topf et al., Gene Ther. 5:507-513(1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and w2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingnucleases and/or donor constructs can also be administered directly toan organism for transduction of cells in vivo. Alternatively, naked DNAcan be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Vectors suitable for introduction of polynucleotides (e.g.nuclease-encoding polynucleotides and/or donor transgenepolynucleotides) described herein include non-integrating lentivirusvectors (IDLV). See, for example, Ory et al. (1996) Proc. Natl. Acad.Sci. USA 93:11382-11388; Dull et al. (1998) J. Virol. 72:8463-8471;Zuffery et al. (1998) J. Virol. 72:9873 -9880; Follenzi et al. (2000)Nature Genetics 25:217-222; U.S. Patent Publication No 2009/054985.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

It will be apparent that the nuclease-encoding sequences and donorconstructs can be delivered using the same or different systems. Forexample, the nucleases and donors can be carried by the same vector.Alternatively, a donor polynucleotide can be carried by a plasmid, whilethe one or more nucleases can be carried by an AAV vector. Furthermore,the different vectors can be administered by the same or differentroutes (intramuscular injection, tail vein injection, other intravenousinjection, intraperitoneal administration and/or intramuscularinjection). In the methods described herein, the vectors areadministered sequentially, typically by first administering thenuclease(s) and subsequently administering the transgene. Multipleadministrations of nuclease(s) and/or transgenes may be conducted.

Thus, the instant disclosure includes in vivo or ex vivo treatment ofany disorder in which a protein is aberrantly expressed. Non-limitingexamples of disease that can be treated include hemophilias (e.g., vianuclease-mediated integration of F7, F8, F9 and/or F10), lysosomalstorage diseases, metabolic diseases, hemoglobinopathies, and othergenetic diseases. See, e.g., See, e.g., U.S. Publication Nos.20120128635; 20140093913; 20140080216 and 20140155468.

The compositions are administered to a human patient in an amounteffective to obtain the desired concentration of the therapeuticpolypeptide in the serum, the liver or the target cells. Administrationcan be by any means in which the polynucleotides are delivered to thedesired target cells. For example, both in vivo and ex vivo methods arecontemplated. Intravenous injection to the portal vein is a preferredmethod of administration. Other in vivo administration modes include,for example, direct injection into the lobes of the liver or the biliaryduct and intravenous injection distal to the liver, including throughthe hepatic artery, direct injection in to the liver parenchyma,injection via the hepatic artery, and/or retrograde injection throughthe biliary tree Ex vivo modes of administration include transduction invitro of resected hepatocytes or other cells of the liver, followed byinfusion of the transduced, resected hepatocytes back into the portalvasculature, liver parenchyma or biliary tree of the human patient, seee.g., Grossman et al., (1994) Nature Genetics, 6:335-341.

The effective amount of nuclease(s) and donor to be administered willvary from patient to patient and according to the therapeuticpolypeptide of interest. Accordingly, effective amounts are bestdetermined by the physician administering the compositions andappropriate dosages can be determined readily by one of ordinary skillin the art. After allowing sufficient time for integration andexpression (typically 4-15 days, for example), analysis of the serum orother tissue levels of the therapeutic polypeptide and comparison to theinitial level prior to administration will determine whether the amountbeing administered is too low, within the right range or too high.Suitable regimes for initial and subsequent administrations are alsovariable, but are typified by an initial administration followed bysubsequent administrations if necessary. Subsequent administrations maybe administered at variable intervals, ranging from daily to annually toevery several years. One of skill in the art will appreciate thatappropriate immunosuppressive techniques may be recommended to avoidinhibition or blockage of transduction by immunosuppression of thedelivery vectors, see e.g., Vilquin et al., (1995) Human Gene Ther.,6:1391-1401.

Formulations for both ex vivo and in vivo administrations includesuspensions in liquid or emulsified liquids. The active ingredientsoften are mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like, andcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances, such as, wetting or emulsifying agents,pH buffering agents, stabilizing agents or other reagents that enhancethe effectiveness of the pharmaceutical composition.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises a zinc finger nuclease (ZFN).It will be appreciated that this is for purposes of exemplification onlyand that other nucleases can be used, for instance homing endonucleases(meganucleases) with engineered DNA-binding domains and/or fusions ofnaturally occurring of engineered homing endonucleases (meganucleases)DNA-binding domains and heterologous cleavage domains, TALENs(TAL-effector DNA binding domains and a restriction endonuclease ormeganuclease domains), Ttago nuclease and/or a CRISPR/Cas systemcomprising an engineered single guide RNA.

EXAMPLES Example 1: Optimization of the Order of Addition ofZFN-Nuclease (mrna) and Donor-AAV in Human Primary Hepatocytes

In order to identify the optimal timing of AAV Donor addition relativeto transfection with mRNAs encoding ZFN in vitro, human primaryhepatocytes (Celsis) were used. For all hepatocyte cultures, thefollowing methods and conditions were used. 24 or 48-well cell culturedishes (VWR) were used which were coated with a mixture of 250 ulmatrigel (BD Biosciences) in 10 ml hepatocytes basal medium, HBM(Lonza), each well was covered in 150 μl of the mixture. Plates wereincubated for 1 hour at 37° C. Thawing/plating media was prepared bycombining 18 ml InVitroGRO CP medium (Celsis In Vitro Technologies) and400 ul Torpedo antibiotic mix (Celsis In Vitro Technologies). Once theplates were prepared, the cells (Celsis In Vitro Technologies, malerhesus monkey plateable hepatocytes, or female plateable humanhepatocytes) were transferred from the liquid nitrogen vapor phasedirectly into the 37° C. water bath. The vial was stirred gently untilthe cells were completely thawed.

The cells were transferred directly into a 50 ml conical tube containing5 ml of pre-warmed thawing/plating medium. To transfer cells completely,the vial was washed with 1 ml of thawing/plating medium. The cells werere-suspended by gently swirling the tube. A small aliquot (20 μl) isremoved to perform a cell count and to determine cell viability usingtrypan blue solution, 1:5 (Cellgro). The cells were then centrifuged at75×g for 5 minutes. The supernatant was decanted completely and thecells were resuspended at 1×10e6 cells/ml. The matrigel mixture wasaspirated from the wells and cells were seeded at 2×10e5 cells/well in a48 well dish. Cells were then incubated in 37° C./5% CO2 incubator. Atthe time of transduction/transfection, cells were switch to maintenancemedium HCM (Lonza and HCM™ SingleQuots™).

The hepatocytes for this experiment were seeded on BD Matrigel™ (BDBiosciences) coated 24 well plates (2e5 cells per well) and leftuntreated for 24 hours to recover. Cells were kept in HCM™ (Lonza). 24hours after seeding the cells were exposed to an MOI of 1e5 of AAV2/6particles containing a DNA donor encoding the secreted embryonicalkaline phosphatase (SEAP) reporter gene protein flanked by hALBspecific homology arms. In these experiments, the AAV virus was preparedusing standard HEK293 production protocols (see Matsushita et al (1998)Gene Therapy 5:938-945).

Either on the same day or 24, 48 or 72 hours later, respectively, 1 μgof mRNA encoding the human albumin-specific ZFN pair 35364-ELD-2A-35396-KKR (a single mRNA encoding both nucleases of the 35364/35396 pairseparated by a self-cleaving 2A peptide sequence) was transfected usingLipofectamine® RNAiMAX (Invitrogen). (See US Patent Publication20130177983 for a description of human albumin specific ZFN pair35364/35396).

Supernatants of the hepatocytes were harvested seven days after andmeasured for SEAP activity (SEAP reporter Gene Assay; Roche).

The results showed that transfection of ZFN mRNA is optimal at 24 hoursor 48 hours after addition of AAV donor (FIG. 2). In contrast, deliveryof ZFN Donor on the same day or 72 hours apart was significantly lessefficient.

Example 2: Optimization of Order of Addition of ZFN-Nuclease (mRNA) andDonor-AAV Addition in NHP Primary Hepatocytes

In order to test the optimized conditions from Example 1 using a moreclinically relevant transgene donor, the human Factor 9 (hF9) gene wasutilized as a donor and was tested in NHP (rhesus) primary hepatocytes.The hepatocytes were seeded on matrigel coated 48 well plates (2e5 cellsper well) as described above and left untreated for 24 hours to recover.24 hours after seeding the cells were exposed to an MOI of 3e5 of AAV2/6particles containing the hF9 transgene flanked by rhesus albuminspecific homology arms of 276 nucleotides (left) and 100 nucleotides(right) respectively (see FIG. 3). Either 24 or 48 hours after donor AAVdelivery, two different pairs of single mRNAs (0.5 μg per single ZFNmRNA) encoding either rhesus albumin-specific ZFNs 37804-ELD/43043-KKRor 36806-FokI WT/35396-FokI WT were transfected as described above. (SeeU.S. Patent Publication 20130177983 and U.S. Pat. No. 10,081,661 for adescription of various albumin specific ZFN pairs). In this example, thetwo ZFNs were delivered as separate mRNAs, rather than as one RNAseparated by a 2A fusion peptide as described in Example 1.

Supernatants of the hepatocytes were harvested every day, and those fromdays 4 to 10 (mRNA transfection is day 1) were analyzed by ELISA againstthe human F.IX protein using a primary antibody (Hematologic systems),which can distinguish between rhesus F.IX protein and human F.IX. Theresults showed that transfection of ZFN mRNA 24 hours after addition ofthe AAV carrying the trangene donor was optimal for transgene expression(see FIG. 3C).

These results are consistent with the observed ZFN expression profile inNHP hepatocytes which peaked between 9-24 hours after ZFN-encoding mRNAtransfection using the 37804/43043 pair as detected by Western analysis(FIG. 4). ZFN expression was monitored through standard Western blotprotocols, using an anti-Fok I primary antibody.

Example 3: Optimization of ZFN-Nuclease (AAV2/6) and hF9 -Donor (AAV2/6)Addition in NHP Hepatocytes

An in vitro AAV transduction system where both ZFN and donor weredelivered to the cells using AAV2/6 viral vectors was investigated tosee if delayed addition of ZFN-AAV and donor-AAV is also superior tosame day addition. Both ZFN and Donors were delivered to NHP (rhesus)primary hepatocytes, where the cells were prepared as described above,and the AAV2/6 (either the ZFN-AAV or the donor-AAV) was introduced tothe cells by adding viral stock pre-diluted in medium. The virus waskept on the cells for 24 hours and then hepatocyte medium was exchanged.Further, supernatants were harvested daily and tested for hF.IXsecretion by ELISA using a primary antibody (Hematologic systems) whichcan distinguish between rhesus F.IX protein and human F.IX.

For these experiments, NHP (rhesus) primary hepatocytes were transducedwith AAV2/6 viruses containing one of the two primate albumin-specificZFNs 36806 or 35396 and AAV2/6 virus comprising the donor (the hF9transgene with primate albumin homology arms). The two virus types wereeither introduced on the same day or the ZFN-AAV was delivered first andthen the donor-AAV was delivered 7, 24 or 48 hours later. The ratio ofdonor-AAV to ZFN-AAV was 5:1 with resulting multiplicities of infection(MOIs) of 3e5 per single ZFN-AAV (total ZFN MOI: 6e5) and 3e6 for thedonor-AAV.

The results showed that the hF.IX expression detected in the cellsupernatant during all time points collected was highest when thedonor-AAV was delivered 24 hr after the ZFN-AAV (see FIG. 5A).

The ratios of the donor-AAV and the ZFN-AAV were then varied anddelivered as before at either the same time, the donor-AAV was deliveredat 7, 24 or 48 hours following the ZFN-AAV. Similar to the previousresult, delivery of the AAV-donor 24 hours after the ZFN-AAV gavesuccessful transgene expression (see FIG. 5B) in this experiment.

Example 4: Optimization of ZFN-Nuclease (AAV2/6) and hF9 -Donor (AAV2/6)Addition in NHP Hepatocytes Via End-Capture

In Example 3, the hF9 transgene was flanked by homology arms withhomology to region surrounding the ZFN cleavage site in the rhesusalbumin gene, allowing the transgene to be integrated either viahomology directed pair or by NHEJ-dependent end capture. A donor wasthen designed with homology arms that are not homologous to the rhesusalbumin gene (the homology arms are homologous to the human F9 gene).Integration of this donor then would be required to occur throughNHEJ-dependent end capture only.

Experimental conditions were those described in Example 3 where theZFN-AAV were added and then the donor-AAV was added either immediatelyor 7, 24 or 48 hours later. Similar to above, cell supernatant wascollected and analyzed for hF.IX protein expression. The results (seeFIG. 6) demonstrated that the delay of 24 hours prior to donor AAVtransfection was optimal for maximal transgene expression independentlyof the integration mechanism.

Example 5: Optimization of ZFN-Nuclease (AAV2/6) and hF9-Donor (AAV2/6)Addition in NHP Hepatocytes

Based on the results from the previous examples, it appears thatincreased transgene (e.g., hF.IX) expression is tightly linked tonuclease (ZFN) expression and activity. Therefore the optimizedconditions of ZFN delivery first and then delivery of the donorcontaining AAV 24 hours later may have led to increased ZFN activity inthe absence of the donor AAV for the first 24 hours, which then drivesincreased donor integration/transgene expression (hF.IX secretion).

To test this, NHP (rhesus) primary hepatocytes were treated with arhesus albumin-specific ZFN pair (37804:43043) and a hF9 transgene donorflanked by homology arms that are homologous to the human albumin locus,meaning that integration of the hF9 transgene can only occur throughNHEJ-dependent end capture. Different Donor:ZFN1:ZFN2 ratios, (2:1:1,6:1:1 and 10:1:1) were also examined to observe the impact of ZFN:Donorratio on both ZFN activity and Donor integration. In these experimentsthe MOI of AAV2/6 encoding the ZFN was fixed at 3e5 per single ZFN(total ZFN MOI: 6e5) and therefore the AAV2/6 Donor MOIs for the otherconditions were 6e5 (2:1:1), 1.8e6 (6:1:1) and 3e6 (10:1:1),respectively.

NHP (rhesus) primary hepatocytes (Celsis) were seeded as describedabove, and both ZFN and donor comprising AAV were delivered as describedabove.

The experiment was carried out in two plates in parallel. The firstplate was harvested four days after ZFN addition to extract genomic DNA(using Qiagen QIAamp DNA micro kit) and analyzed for ZFN activity asfollows. Briefly, the region comprising the cleavage site was amplifiedby PCR, and following amplification, the PCR product was sequenced viaMiSeq high throughput sequencing analysis according to manufacturer'sinstructions (Ilumina).

For the second plate, duplicate experimental conditions as those for thefirst plate were used and supernatants were collected at three timepoints: 2 days after ZFN addition, 5 days after ZFN addition (where thesupernatants from days 3-5 were combined) and 8 days after ZFN addition(where the supernatants from days 6-8 were combined). These supernatantsthen were tested for secreted hF.IX protein using a primary antibody(Hematologic systems) which can distinguish between rhesus F.IX proteinand human F.IX protein. Additionally, cells were harvested at Day 8 toextract genomic DNA for ZFN activity analysis by sequencing as describedabove.

The results showed that same day transduction of AAV-ZFN and AAV-donorlead to decreased ZFN activity compared to transduction with ZFN alone.The sequencing analysis detected less indels on days 4 and 8 in allZFN:donor ratios in the samples where the ZFN and donor-AAV particleswere transduced on the same day (see FIG. 7). In contrast, when theAAV-ZFN was added first followed by the AAV-donor 24 hours later, theZFN activity (% indels) was identical with the ZFN only transfectionsample, irrespective of the ZFN:Donor ratio.

As expected the ZFN activity also correlated with hF.IX proteinsecretion, indicative of hF9 transgene integration, as detected byELISA. As before optimized delivery conditions lead to a more than 2fold increase of hF.IX secretion irrespective of the ZFN:Donor ratio.

Example 6: In Vivo Testing of Staggered ZFN-Nuclease (AAV2/8) and hF9-Donor (AAV2/8) Addition in Mice

In order to test whether the optimized AAV-ZFN/AAV-donor additionconditions can also be used in vivo, several addition strategies weretested side-by-side in mice using the mouse albumin-specific ZFNs30725:30724 and a hF9 transgene donor that was flanked with homologyarms with homology to the mouse albumin gene surrounding the ZFNcleavage site (ZFNs 30724 and 20725 as described in U.S. PatentPublication 20130177983 with engineered cleavage domains).

C57/B16 mice (cohorts of five) were injected with AAV2/8 encoding either1.5 ell (total) viral genomes (VGs) of AAV-ZFN only, or 9e11 (total)AAV-donor plus AAV-ZFN, which represents a Donor: ZFN ratio of 3:1. Forthis study either the donor-AAV was delivered first and then the ZFN-AAVwas delivered 24 hours later (group 1); both ZFN-AAV and donor-AAV wereadministered at the same time (group 2); ZFN-AAV was delivered first andthen the donor-AAV was delivered 24 hours later, (group 3); or ZFN-AAVwas delivered first, and the donor-AAV was delivered 72 hr later (group4); or ZFN-AAV was delivered first, and the donor-AAV was delivered 120hours later (group 5). In these studies, both were delivered byinjection to the tail vein as described in U.S. Patent Publication No.20120128635. Seven days after ZFN delivery in all groups, serial bleedof the different groups was carried out for analysis of hF.IX secretioninto the plasma.

ELISA for human hF.IX (Affinity) performed on day seven revealed thataddition of donor-AAV first was indistinguishable from addition ofZFN-AAV and donor-AAV on the same day (see FIG. 8). In contrast, if thedonor-AAV was administered 24 hours or 72 hours after the ZFN-AAV,levels of hF.IX in the plasma were two to three fold higher respectivelythan the administration on the same day. In contrast, administration ofthe Donor 120 hours after the ZFN resulted in complete lack of hF9expression. Probability analysis (Student's T test) demonstrated asignificant difference between samples from the mice that got the donorand ZFN on the same day, or got the donor 24 hours ahead of time ascompared to mice that got the donor-AAV three days after they hadreceived the ZFN-AAV.

Analysis of genomic DNA from liver tissue from a satellite mouse groupalso sacrificed on day seven showed that the higher hF.IX expressioncorrelated with the higher levels of albumin gene modification, whichwas two-fold higher when the donor-AAV was administered 3 days after theZFN-AAV (see FIG. 9A). Additionally, the levels of targeted genemodification observed for the mice that had received the donor-AAV 3days following the ZFN -AAV were similar to the group that only receivedZFN-AAV. This was consistent with the in vitro results shown in Example5. However, transduction efficiency (assayed by detection of VGs perdiploid genome) with ZFN-AAV was the same for all groups but ZFNexpression levels, as analyzed by standard Western blot analysis, werehigher when either the ZFN-AAV was delivered alone or when the donor-AAVwas delivered three days after the ZFN-AAV (FIG. 9B) in comparison towhen the donor-AAV was given prior to, or at the same time as theZFN-AAV.

When the different dosing cohorts are analyzed for serum hF.IX levelsover time, long term expression of the transgene is observed (FIG. 10).Transgene expression can be detected even at 77 days after transductionwith the AAV-ZFN. The groups all achieve appreciable transgeneexpression, but transduction of the AAV-ZFN three days before additionof the AAV-donor virus achieves increased expression the fastest.

Example 7: In Vivo Testing of Staggered ZFN-Nuclease (AAV2/8) and hF9-Donor (AAV2/8.2) Addition in NHP

In order to test whether the optimized AAV ZFN/AAV Donor additionconditions can also be used in NHP in vivo, studies are performed usingseveral different addition strategies in rhesus monkeys using the rhesusalbumin-specific ZFNs 37804:43043 and a hF9 transgene donor that isflanked with homology arms with homology to the rhesus albumin genesurrounding the ZFN cleavage site. In these experiments, the ZFN-AAVeach contain a single ZFN coding sequence, so to deliver the ZFN pair,two ZFN-AAV particle types are given. The ZFN-AAV virus particlestogether are given in a 8:1:1 ratio (Donor:ZFN1:ZFN2) with the AAV-donorparticles, and pairs of monkeys are given the ZFN-AAV and donor-AAVeither on the same day, or given ZFN-AAV 1 day or 3 days prior to thedonor-AAV. Serial bleeds are performed over time to test for serumhF.IX.

The results demonstrated that delay of 1 to 3 days between when theanimals receive the ZFN-AAV and the donor-AAV gave the most rapidexpression of the transgene up until Day 28 (FIG. 11A). Analysis of thehF.IX peak levels of all animals during study duration (>30 weeks)revealed that of the 5 animals expressing detectable levels of h.FIX the3 highest expressing animals had been treated with AAV-donor particleseither 1 or 2 days after the AAV-ZFN particles (FIG. 11B).

Taken together, these data show that separate administration ofnucleases and donor transgenes showed significant enhancement oftransgene expression (3-fold as compared to same day administration).Thus, nuclease mediated integration of transgenes can be enhanced byserial administration of nuclease(s) and transgenes when both aredelivered as viral particles or mRNA, with a delay of hours to daysbetween the administrations.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A method of integrating one or more transgenesinto a genome of an isolated cell, the method comprising: introducing,into the cell, (a) an adeno-associated type virus (AAV) donor vectorcomprising the one or more transgenes; and (b) a zinc finger nuclease(ZFN) that cleaves an endogenous gene, wherein the ZFN comprises firstand second ZFNs and further wherein the ZFN is introduced before thedonor vector, such that the one or more transgenes are integrated intothe cleaved endogenous gene.
 2. The method of claim 1, wherein the atleast one nuclease is introduced at least 4 hours before the AAV donorvector.
 3. The method of claim 1, wherein the first and second ZFNs ofthe ZFN are administered as RNA.
 4. The method of claim 3, wherein theRNA is mRNA.
 5. The method of claim 1, wherein the first and second ZFNsof the ZFN are administered using one or more AAV vectors.
 6. The methodof claim 5, wherein the endogenous gene is a safe-harbor locus.
 7. Amethod of providing a functional protein lacking or deficient in asubject, the method comprising integrating one or more transgenes intoan isolated cell according to the method of claim 1 and administeringthe isolated cell to the subject.
 8. The method of claim 7, wherein thecell is a stem cell.
 9. The method of claim 1, wherein the AAV vectorcomprises an AAV2 vector, an AAV6 vector or chimeric AAV2/6.